Ocena stężenia podjednostki alfa po podaniu TRH: przydatny test w przedoperacyjnej diagnostyce gonadotropinoma?

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1 / ORIGINAL PAPERS Endokrynologia Polska / Polish Journal of Endocrinology Tom/Volume 56; Numer/Number 1/2005 ISSN X Ocena stężenia podjednostki alfa po podaniu TRH: przydatny test w przedoperacyjnej diagnostyce gonadotropinoma? Anna Gruszka 1, Jolanta Kunert-Radek 2, Marek Pawlikowski 1 1 Zakład Endokrynologii Doświadczalnej i Diagnostyki Hormonalnej, Instytut Endokrynologii UM w Łodzi 2 Klinika Endokrynologii, Instytut Endokrynologii UM w Łodzi Streszczenie Wstęp: Klinicznie nieczynne gruczolaki przysadki (clinically nonfunctioning pituitary adenomas, CNFPAs) stanowią około 30% makrogruczolaków przysadki i są najczęściej guzami typu gonadotropinoma. Celem pracy była weryfikacja użyteczności pomiaru stężenia podjednostki alfa hormonów glikoproteinowych (α-su) w surowicy w odpowiedzi na pobudzenie egzogennym TRH w rozpoznawaniu gonadotropowego charakteru CNFPAs przed leczeniem neurochirurgicznym. Materiał i metody: Badania przeprowadzono u 14 pacjentów z klinicznie nieczynnymi gruczolakami przysadki. U każdego chorego przed zabiegiem chirurgicznym oceniono stężenie α-su w surowicy w odpowiedzi na podanie TRH. Jako odpowiedź znamienną przyjęto arbitralnie wzrost stężenia α-su o ponad 50% w stosunku do wartości wyjściowej. Wyniki: Pacjenci zostali podzieleni na dwie grupy, po 7 chorych każda. Grupę pierwszą stanowili pacjenci z guzami typu gonadotropinoma (guzy immunopozytywne dla FSH i/lub LH bądź ich wolnych podjednostek). W grupie drugiej znaleźli się chorzy z gruczolakami immunonegatywnymi dla gonadotropin i α-su. Podstawowe stężenie α-su powyżej normy stwierdzono u dwóch pacjentów w grupie pierwszej (gruczolaki gonadotropowe) oraz u jednego w grupie drugiej. Wzrost stężenia α-su po podaniu TRH o ponad 50% obserwowano u wszystkich z wyjątkiem jednego badanego w grupie pierwszej oraz u żadnego z siedmiu chorych z guzami immunonegatywnymi dla FSH, LH lub α-su. U trzech z siedmiu pacjentów z gruczolakami immunonegatywnymi dla gonadotropin stwierdzono obniżenie stężenia α-su w surowicy po podaniu TRH. Wniosek: Pomiar stężenia α-su w odpowiedzi na podanie TRH może być przydatny w przedoperacyjnej identyfikacji guzów gonadotropowych wśród innych nieczynnych hormonalnie gruczolaków przysadki. (Endokrynol Pol 2005; 1(56): 14-18) Słowa kluczowe: podjednostka α, test z TRH, guzy przysadki, gonadotropinoma * Prof. dr hab. med. M. Pawlikowski Zakład Endokrynologii Doświadczalnej i Diagnostyki Hormonalnej, Instytut Endokrynologii Uniwersytetu Medycznego w Łodzi ul. dr Sterlinga 3, Łódź tel/fax: pawlikowski.m@wp.pl 14

2 / ORIGINAL PAPERS Endokrynologia Polska / Polish Journal of Endocrinology Tom/Volume 56; Numer/Number 1/2005 ISSN X Serum alpha-subunit elevation after TRH administration: a valuable test in presurgical diagnosis of gonadotropinoma? Anna Gruszka 1, Jolanta Kunert-Radek 2, Marek Pawlikowski 1 1 Dept. of Experimental Endocrinology and Hormone Diagnostics, Institute of Endocrinology, Medical University of Łódź, Poland 2 Dept. of Endocrinology, Institute of Endocrinology, Medical University of Łódź, Poland Abstract Objectives: Clinically nonfunctioning pituitary adenomas (CNFPAs) represent about 30% of pituitary macroadenomas, gonadotropinomas being the most frequent among them. The aim of the present study is to re-evaluate the usefulness of the measurement of α-su serum level in response to TRH stimulation in detecting the gonadotropic nature of nonfunctioning pituitary adenomas before the neurosurgical treatment. Material and methods: We have studied 14 patients with CNFPAs. The response of α-su to the administration of TRH was studied in each patient before the surgery. α-su blood serum level increase over 50% of the baseline level after TRH treatment was considered to be significant. Results: The patients were divided into 2 groups, each including 7 subjects. The first group included the patients with gonadotropinomas (tumors immunopositive for FSH and/or LH or their free subunits). The second group included the patients with adenomas immunonegative for gonadotropins and α-su. The basal level of α-su was elevated over the upper limit of normal range in two patients of the first group (gonadotroph adenomas) and in one in the second group. All but one patient from the first group and none of seven patients with tumors immunonegative for FSH, LH or α-su, had a significant α- SU (over 50%) response to TRH. In three of seven patients with gonadotropins immunonegative tumors a decrease of α-su serum level after TRH was observed. Conclusion: The measurement of α-su serum level in response to TRH administration seems to be useful in preoperative identification of gonadotroph adenomas among other nonfunctioning pituitary adenomas. (Pol J Endocrinol 2005; 1(56): 14-18) Key words: α subunit; TRH test; pituitary tumors; gonadotropinoma * Prof. dr hab. med. M. Pawlikowski Dept. of Experimental Endocrinology and Hormone Diagnostics, Institute of Endocrinology, Medical University of Lodz Dr Sterling str. 3, Lodz, Poland tel/fax: pawlikowski.m@wp.pl Acknowledgements This study was supported by a grant from the State Committee for Scientific Research of Poland No 3PO5B (for JKR). 15

3 TRH test in diagnosis of gonadotropinoma Gruszka A. Introduction Clinically nonfunctioning pituitary adenomas (CNFPAs) represent about 30% of surgically removed pituitary macroadenomas. Usually, these tumors grow undetected until they become large enough to cause mass effects such as visual impairment, headaches and/or symptoms of hypopituitarism [6, 7, 15]. In spite of no clinical features of pituitary hormones hypersecretion, the presence of pituitary hormones is detected in most of CNFPAs in postoperative immunohistochemical investigations. It has been found that the majority of these tumors (50-75%) show positive immunostaining for gonadotropins and/or their free subunits implying a gonadotroph cell origin [1, 8, 11]. These results are in agreement with data from in vitro cell cultures showing gonadotropins secretion in most of CNFPAs [3, 9]. These findings have been also supported by in situ hybridization studies [5]. Gonadotroph adenomas are rarely associated with the increased basal plasma levels of luteinizing hormone (LH) and/or follicle-stimulating hormone (FSH). The elevated free α subunit (α-su) concentrations occur only in half of the patients. We have been still looking for a diagnostic tool allowing preoperative identification of gonadotroph adenomas among other nonfunctioning pituitary adenomas. Paradoxical responses of LH, FSH and their free subunits to the intravenous administration of thyrotropinreleasing hormone (TRH) have been previously reported in patients with CNFPAs but their diagnostic value is considered as rather limited [2, 4, 10, 13, 14, 16]. The abnormal response of free α-su to TRH was reported in approximately 30% of patients only [13, 14] and free β-lh response ranged from 26% [2] to 77,8% [16] of patients with CNFPAs. The aim of the present study is to re-evaluate the usefulness of the measurement of α-su serum level in response to TRH stimulation in detecting the gonadotropic nature of nonfunctioning pituitary adenomas before the neurosurgical treatment. Materials and Methods The study was carried out in 14 patients with pituitary macroadenomas diagnosed before surgery as nonfunctioning. The average age of the patients (9 men and 5 women) was 53.8 yrs. All the patients underwent radiological (MRI) and hormonal examinations including baseline PRL, GH, IGF-1, ACTH, cortisol, LH, FSH, α-su, TSH, ft 3, ft 4, testosterone (in men) or estradiol (in women). None of the patients had clinical symptoms of hormonal hypersecretion of pituitary hormones and their blood levels were within the normal ranges. In every patient the histological and immunohistochemical examinations were performed after surgical excision of the pituitary tumor. Tumors were fixed in Bouin-Holland fixative and paraffin sections were stained with Herlant s tetrachrome and immunostained with antisera against pituitary hormones or α-su. The following antisera were used: polyclonal anti-human PRL (Dako, Denmark), polyclonal anti-human GH (Dako or Immunon, USA), monoclonal anti-human LH (Dako) or polyclonal (Immunon), monoclonal anti-human FSH (Dako), monoclonal anti-human TSH (Immunotech, France), monoclonal anti-human α-su (Immunotech) and polyclonal anti-acth (Sigma, USA). The response of α-su to the administration of TRH was studied in each patient before the surgery. The blood samples were collected before, 30 and 60 minutes after the intravenous injection of 200 mg TRH. α-su blood serum level increase over 50% of the baseline level after TRH treatment was arbitrarily considered to be significant. The data were analyzed statistically using χ 2 test. Results The patients were divided into 2 groups, each including 7 subjects. The first group included the patients with tumor immunopositivity for FSH and/or LH or their free subunits. These tumors we classified as gonadotropinomas. Four tumors from this group exhibited also the positive immunoreaction for PRL, in spite of the normal PRL serum levels, and 5 tumors also an immunopositivity for GH in spite of the normal level of GH and no symptoms of acromegaly. In one case, the tumor was also immunopositive for ACTH, in spite of no symptoms of Cushing s disease. The second group included the patients with adenomas immunonegative for gonadotropins and α-su. One tumor showed the immunopositivity for ACTH, two for GH and 2 for PRL. Only in 2 cases the pituitary adenomas were totally devoid of hormone immuno-expression (see table Ia and Ib). The basal level of α-su was elevated over the upper limit of normal range in two patients of the first group (gonadotroph adenomas) and in one in the second group. All but one patient from the first group and none of seven patients with tumors immunonegative for FSH, LH or α-su, had a significant α-su (over 50%) response to TRH (table Ia and Ib). In three of seven patients with gonadotropins immunonegative tumors a decrease of α-su serum level after TRH was observed. Discussion Our findings confirm the earlier data that clinically nonfunctioning pituitary adenomas express in majority the positive immunoreactions to pituitary hormones and/or their free subunits in spite of their normal blood levels and lack of symptoms of pituitary hyperfunction [8]. The most frequently 16

4 Endokrynologia Polska / Polish Journal of Endocrinology 2005; 1 (56) Table Ia and Ib The results of immunohistochemical investigation and serum α-subunit (α-su) level at baseline and in response to TRH in patients with clinically nonfunctioning pituitary macroadenomas. Ia. Tumors immunopositive for gonadotropins or their free subunits, Ib. Tumors immunonegative for gonadotropins or their free subunits. The normal (N) α-su values in men: miu/ml, in premenopausal women: 0 0,9 miu/ml, in postmenopausal women: miu/ml % increase after TRH = (maximal response to TRH mean basal value) (100/mean basal value). A response greater than 50% was considered significant (S). NS non significant. Tabela Ia i Ib Wyniki badania immunohistochemicznego oraz stężenie podjednostki α (α-su) w warunkach podstawowych i w odpowiedzi na TRH u pacjentów z klinicznie nieczynnymi gruczolakami przysadki. Ia. Guzy immunopozytywne dla gonadotropin bądź ich wolnych podjednostek, Ib. Guzy immunonegatywne dla gonadotropin bądź ich wolnych podjednostek. Prawidłowe stężenie α-su w surowicy u mężczyzn: 0-0,8 miu/ml, u kobiet przed menopauzą: 0 0,9 miu/ml, po menopauzie: 0-1,6 miu/ml. % wzrostu po TRH = (maksymalny wzrost po TRH średne stężenie podstawowe) (100/ średne stężenie podstawowe). Wzrost stężenia α-su o ponad 50% w stosunku do wartości wyjściowej przyjęto za znamienny statystycznie (S). NS nieznamienny. Table Ia Patient Sex Age [yrs] IHC FSH LH α-su ACTH PRL GH TSH basal α -SU serum level [miu/ml] after TRH % increase administration after TRH G.R. M (N) ( ) S J.T. M (N) (N) S J.W. M (N) (N) 64.9 S K.J. F ( ) ( ) 22.1 NS K.M. M ( ) ( ) S M.C. M (N) ( ) S W.L. M (N) ( ) S Table Ib Patient Sex Age [yrs] IHC FSH LH α-su ACTH PRL GH TSH basal α -SU serum level [miu/ml] after TRH % increase administration after TRH C.J. M (N) 0.37 (N) NS G.B. F (N) 0.64 (N) NS K.J. F (N) 0.59 (N) 28.3 NS K.Z. F ( ) 0.27 (N) NS M.H. M (N) 0.35 (N) 34.6 NS S.J. M (N) 1.61 ( ) 46.3 NS W.M. F (N) 0.34 (N) 47.8 NS the immunopositivity for gonadotropins or their free subunits is observed. We have shown that the frequency of recurrence is higher in tumors expressing gonadotropins [8] and these tumors present the highest values of proliferation markers [12]. Therefore it is important to search for the markers allowing the detection of these tumors before the neurosurgical treatment. In the present paper we have shown that α-su measurement after TRH administration is a good diagnostic procedure to discriminate the gonadotropinomas from the other CNFPAs before surgery, providing that we consider as abnormal α-su response higher than 50% over the basal level. Nobels et al. [10] proposed an increase of the α-su over 30% as the limit of the abnormal response. When we analyzed our data according to this criterium, we had to consider 6/7 tests in gonadotropinoma group as abnormal. However, 3/7 tests in the group of gonadotropin-immunonegative tumors could be considered as abnormal, too. Thus, the test interpreted with this criterium did not differentiate gonadotropin-expressing and nonexpressing tumors. It can be noticed that 5 adenomas in the gonadotropinoma group co-expressed also GH (in spite of the lack of symptoms of acromegaly). A question had arisen whether this co-expression could be responsible for the abnormal response to TRH. Although GH-secreting tumors in acromegalic patients often present the abnormal response of 17

5 TRH test in diagnosis of gonadotropinoma Gruszka A. GH to TRH stimulation, such a presumption seems unlikely. The exaggerated response of α-su occurred also in monohormonal gonadotropinomas not expressing GH. On the other hand, in two monohormonal silent somatotropinomas included in the second group the administration of TRH failed to stimulate the α-su level. Although our observations are based on the limited number of patients, they encourage to use the α-su measurement after TRH administration in presurgical diagnosis of gonadotropinomas. References 1. Chaidarun SS, Klibanski A. Gonadotropinimas. Semin Reprod Med 2002; 20: Chanson P, Pantel J, Young J et al. Free luteinizing hormone beta subunit in normal subjects and patients with pituitary adenomas. J Clin Endocrinol Metab 1997; 82: Croue A, Beldent V, Rousselet MC et al. Contribution of immunohistochemistry, electron microscopy and cell culture to the characterization of nonfunctioning pituitary adenomas: a study of 40 cases. Hum Pathol 1992; 23: Gil del Alamo P, Pettersson KS, Saccomano K et al. Abnormal response of luteinizing hormone beta subunit to thyrotropinreleasing hormone in patients with non-functioning pituitary adenoma. Clin Endocrinol (Oxf) 1994; 41: Jameson JL, Klibanski A, Black PM et al. Glycoprotein hormone genes are expressed in clinically nonfunctioning pituitary adenomas. J Clin Invest 1987; 80: Katznelson L, Alexander JM, Klibanski A. Clinically nonfunctioning pituitary adenomas. J Clin Endocrinol Metab 1993; 76: Kreisman SH, Jackson IMD. Gonadotroph adenomas. Endocrinologist 1999; 9: Kunert-Radek J, Radek A, Gruszka A, Pawlikowski M. Immunohistochemical investigation of clinically nonfunctioning pituitary tumors as a prognostic factor of tumor recurrence. Proceedings of the 12 th Congress of the International Society of Endocrinology; Aug 31 th Sept 4 th 2004; Lisbon, Portugal: Medimond, Bologna, Italy; p Kwekkeboom DJ, de Jong FH, Lamberts SWJ. Gonadotropin release by clinically nonfunctioning and gonadotroph pituitary adenomas in vivo and in vitro: relation to sex and effects of TRH, GnRH and bromocriptine. J Clin Endocrinol Metab 1989; 68: Nobels FR, Kwekkeboom DJ, Coopmans W et al. A comparison between the diagnostic value of gonadotropins, alpha-subunit and chromogranin A and their response to thyrotropin-releasing hormone in clinically nonfunctioning, alpha-subunit-secreting and gonadotroph pituitary adenomas. J Clin Endocrinol Metab 1993; 77: Pawlikowski M, Kunert-Radek J, Radek A. Gonadotropinoma a frequent subtype of a pituitary adenoma. Endocrinol Pol 2000; 51: Pawlikowski M. Immunocytochemical prognostic markers in pituitary tumors. Folia Histochem Cytobiol 2001; 39: Popovic V, Damjanovic S. The effect of thyrotropin-releasing hormone on gonadotropin and free alpha-subunit secretion in patients with acromegaly and functionless pituitary tumors. Thyroid 1998; 8: Samejima N, Yamada S, Takada K et al. Serum alpha-subunit levels in patients with pituitary adenomas. Clin Endocrinol (Oxf) 2001; 54: Snyder PJ. Gonadotroph adenomas. J Clin Endocrinol Metab 1995; 80; Somjen D, Tordjman K, Kohen F et al. Combined beta FSH and beta LH response to TRH in patients with clinically nonfunctioning pituitary adenomas. Clin Endocrinol (Oxf) 1997; 46: